The introduction of an exogenous nucleic acid sequences (e.g., DNA) into cells plays a major role in a variety of biotechnology and related applications, including research, synthetic and therapeutic applications. Research applications include the production of transgenic cells and animals. Synthetic applications include the production of peptides and proteins, as well as therapeutic RNAs, such as RNAi reagents, Cas9 guide RNAs, or ribozymes. Therapeutic applications include gene therapy applications.
In many of these applications, it is desirable to introduce the exogenous DNA in a manner such that it provides for long-term transgene expression, i.e., long-term expression of the protein or RNA encoded by the exogenous DNA. Plasmid DNA based expression vectors have become an essential tool for both biological discovery and the development of new therapeutics. However, the inability to achieve sustained elevated levels of transgene expression in vivo have limited their usage. Standard plasmid vectors composed of (i) a transgene expression cassette (which expresses a transgene, e.g., in eukaryotic cells), and (ii) plasmid bacterial backbone (BB) sequences (which are generally thought of as non-transcribed spacer sequences in the context of transgene expression in eukaryotic cells), are able to express a high level of transgene product shortly after entering eukaryotic cells (e.g., mammalian cells), but the transgene product usually declines to very low or undetectable levels in a period of days even though vector DNA is still present.
To overcome plasmid DNA related transgene silencing in vivo, minicircle DNA vectors have been developed by removing the plasmid bacterial backbone (BB) from the plasmid vector (e.g., via homologous recombination). Minicircle DNA vectors persistently express transgenes at 10- to 1,000-fold levels compared to what can be obtained from a conventional plasmid in quiescent tissues in vivo. Because the plasmid BB, which contains the bacterial replication origin and a selectable marker, has been removed from a minicircle DNA vector, the conventional plasmid preparation method is not suitable for minicircle DNA preparation. Even robust methods that have been developed to produce minicircle DNA vectors are more complicated than conventional plasmid preparation, which limits the usage of minicircle DNA vectors.
Expression vectors that can provide for sustained elevated levels of transgene expression, and that can be produced using simple well-established methods are needed in the art. The present disclosure provides compositions and methods that address these issues.